As described above, liposomes are utilized in a wide variety of fields. However, it was impossible by the prior-art methods for preparing liposomes to produce liposomes with a high-viscosity aqueous solution innerly incorporated. Prior methods for preparing liposomes include so-called film methods, detergent-removal methods and reverse-phase evaporation methods. The film methods comprises forming a dried thin film of liposome-forming lipids on the inner surface of a vessel, to which an aqueous solution of a substance to be incorporated is added and subjecting the resulting mass to shaking or ultrasonication. The detergent-removal method depends upon removing detergents by dialysis from an aqueous solution which contains detergents and phospholipids to form mixed micelles, which results in the formation of liposomes. The reverse-phase evaporation method is a method in which liposomes are prepared by adding to an organic solvent solution of liposome-forming lipids an aqueous solution of a substance to be incorporated to form a water-in-oil emulsion and then removing the organic solvent by evaporation. According to these prior art methods, whereas liposomes are formed in cases where the aqueous solution to be innerly incorporated is of a low viscosity, the yield will be extremely low if it is of a high viscosity over 10 cP (20.degree. C.), and desirable liposomes will not be produced in some cases. This has restricted uses of liposomes. For example, liposomes containing an aqueous hemoglobin solution are known as artificial erythrocytes, but, because of the viscosity restriction, the hemoglobin concentration cannot be so high as that of natural hemoglobin (which is 35% (w/v)), being as low as approximately 15%, so that the oxygen-carrying capacity is low.
On the other hand, Miller et al. reported a hemoglobin-containing liposome prepared by the so-called film method (U.S. Pat. No. 4,133,874). According to the method, the liposome is produced by dissolving liposome-forming lipids in an appropriate solvent such as chloroform, distilling off the solvent from the resulting solution to form a film of the lipids, to which an aqueous solution of hemoglobin is added and then subjecting the liposome to ultrasonication. The method is advantageous in that the hemoglobin can be kept with relatively little degradation due to contact with oxygen only for a short period. However, the oxygen-carrying function is likely to be lost due to slow oxidation of the heme iron of hemoglobin in the liposome during storage. Although hemoglobin in the blood cell is provided with a mechanism wherein the hemoglobin oxidized to the methemoglobin is reduced to the original form by the action of enzymes, such mechanism is not workable if removed outside the blood cell by hemolysis so that the oxygen-carrying capacity is lost once denatured to the methemoglobin.
Hemoglobin is a macromolecular substance having a molecular weight of about 65,000 the aqueous solution of which has a high viscosity. According to the prior methods liposomes containing an aqueous solution with such a high molecular weight and viscosity cannot be prepared, and there are obtained liposomes containing hemoglobin in concentration as low as about 15%. As compared with the hemoglobin concentration of about 35% in natural erythrocytes, the above-cited concentration is not high enough for carrying oxygen. In order to provide a oxygen-carrying capacity equal to that of blood, the concentration of the hemosome itself cannot be increased, which will increase the concentration of the membrane-forming lipid material and induce a safety problem. Furthermore, viscosity of the lipid suspension will become so high that the dynamics blood flow will unfavorably be affected. Under such circumstances, liposomes innerly containing hemoglobin in a higher concentration are desirable.
The above-mentioned object is achieved by the present invention a described below.